Gel filtration chromatography is commonly used for the separation of buffer components from macromolecules, such as proteins and DNA. This application is typically referred to as desalting or buffer exchange. It is an essential technique in life science laboratories, where it is used to remove excess salt, metabolites, biotin, crosslinkers, reactive dyes, radioactive labels and drugs, while transitioning the sample of interest into a buffer appropriate for subsequent applications and analysis. There are a number of formats and resins commercially available for performing desalting applications in the laboratory. For sample volumes less than 4mL, gravity-flow (drip) columns and spin-column formats are the most common. Different resins are available having size-exclusion limits ranging from 2000 to 40,000 Daltons. However, the most popular resins for small-scale protein desalting applications are those with nominal exclusion limits corresponding to 5000 to 7000kDa (typically expressed as 5K to 7K MWCO, molecular-weight cutoff).

Figure 1. Summary of available spin desalting column products from three manufacturers. The specified ranges are the manufacturer-recommended sample volumes for spin protocols with each column. Stars indicate columns that were tested in this study.

Results and discussion

The 7K MWCO Zeba Desalting Resin and accompanying sizes of centrifuge-ready column formats were designing to provide excellent gel-filtration performance over a wide range of sample sizes and protein concentrations. To demonstrate this, we tested two sizes (0.5mL and 10mL columns) of Zeba Desalting Columns alongside the closest available sizes of columns from GE Healthcare and Bio-Rad Corporation. Protein recovery data after processing a range of sample volumes and protein concentrations for BSA and HeLa cell lysate samples are summarized in Table 1 (small columns) and Table 2 (large columns).

Desalting columns were equilibrated with a low-salt (25mM NaCl) buffer. Samples were prepared at various concentrations in high-salt (500mM NaCl) buffer and then desalted into the final buffer using each column and method. Columns were used according to respective manufacturers’ recommended protocols. For all columns tested, the efficiency of salt removal was equivalent (>95%, data not shown). Essentially, there were no significant differences in buffer-exchange performance among columns tested, except where the sample volume exceeded what is recommended for a particular column (e.g., 3.5mL samples in PD-10 columns, Table 2). Protein concentration was determined using the BCA Protein Assay Kit (Part No. 23227). Protein recoveries with lysate samples (e.g., HeLa lysate) are typically slightly lower than with pure proteins (e.g., BSA) due to loss of small molecular weight compounds and lysis contaminants that can influence protein assay results.

Table 1. Comparison of protein recoveries with different microcentrifuge desalting columns and conditions. Recoveries are expressed to the nearest 5% values based on several trials. See text for details. Column products are Thermo Scientific Zeba (Thermo Fisher Scientific), SpinTrap G-25 (GE Healthcare), and Micro Bio-Spin 6 (Bio-Rad); see Figure 1.

Zeba
0.5mL

SpinTrap
G-25

Micro
Bio-Spin 6

Sample
(concentration)

Sample
Volume

Recovery

Recovery

Recovery

BSA
0.04mg/mL

40μL

70-80%

<10%

<10%

80μL

80-95%

<10%

<10%

120μL

>90%

<10%

<20%

BSA
0.2mg/mL

40μL

70-80%

<15%

25-35%

80μL

80-95%

35-45%

45-60%

120μL

>90%

55-70%

65-80%

BSA
1mg/mL

40μL

80-95%

45-60%

70-80%

80μL

>90%

60-75%

80-90%

120μL

>90%

75-90%

>90%

HeLa lysate
0.2mg/mL

40μL

45-55%

<10%

<15%

80μL

60-70%

15-25%

30-40%

120μL

70-80%

45-55%

50-60%

HeLa lysate
1mg/mL

40μL

65-75%

45-55%

45-55%

80μL

75-85%

60-70%

70-80%

120μL

80-90%

70-80%

80-90%

Table 2. Comparison of protein recoveries with different centrifuge and gravity-flow (drip) desalting columns and conditions. Recoveries are expressed to the nearest 5% values based on several trials. See text for details. Column products are Thermo Scientific Zeba (Thermo Fisher Scientific) and PD-10 (GE Healthcare); see Figure 1.

Zeba
10mL
Spin

PD-10
(8.3mL)
Spin

PD-10
(8.3mL)
Drip †

Sample
(concentration)

Sample
Volume

Recovery

Recovery

Recovery

BSA
0.04mg/mL

1.5mL

75-95%

20-30%

40-50%

2.5mL

75-95%

30-40%

45-55%

3.5mL

75-95%

[60-70%]

[55-65%]

BSA
0.2mg/mL

1.5mL

>90%

60-70%

>90%

2.5mL

>90%

70-80%

80-95%

3.5mL

>90%

[75-85%]

[75-85%]

BSA
1mg/mL

1.5mL

>90%

80-90%

>90%

2.5mL

>90%

80-90%

>90%

3.5mL

>90%

[80-90%]

[85-95%]

HeLa lysate
0.2mg/mL

1.5mL

70-85%

55-65%

>90%

2.5mL

70-85%

65-75%

80-95%

3.5mL

80-95%

[70-80%]

[75-85%]

HeLa lysate
1mg/mL

1.5mL

80-95%

80-90%

>90%

2.5mL

80-95%

80-95%

>90%

3.5mL

85-95%

[80-95%]

[85-95%]

† In the gravity-flow protocol, samples were recovered in 3.5mL regardless of initial sample volume. Protein recovery was determined on total final volume.
Note: Brackets (3.5mL samples in PD-10 columns) indicate that the sample volume significantly exceeds the maximum recommended for the column. Buffer exchange was incomplete in this condition.

In nearly every condition, protein recovery was highest (i.e., protein loss was lowest) for the Zeba columns compared to the competitor products. Protein recovery was especially poor with the GE Healthcare and Bio-Rad products for low-concentration samples (e.g., BSA at <0.2mg/mL), especially when applied in a small volume (40µL in Table 1; 1.5mL in Table 2). The data in Tables 1 and 2 are more easily visualized and summarized by representing them as surface maps of protein recovery versus sample volume and sample concentration (Figure 2).

Figure 2. Surface maps of protein recovery vs. sample concentration and sample volume. Graphed data are values for the BSA samples from Tables 1 and 2. See text for details.

The high levels of protein loss seen with the GE Healthcare and Bio-Rad spin desalting columns apparently result from a combination of (2) weak non-specific binding of protein to the resin and (1) entrapment of the sample when there is insufficient liquid to carry it through the resin during centrifugation. Non-specific binding is evident in the test conditions involving low-concentration samples, where more than 50% of the sample proteins fail to pass through the column regardless of the volume. Entrapment is evident upon comparison of recovery values for the PD-10 columns processed by centrifugation (spin) versus gravity-flow (drip); when a large volume of buffer is used to push a smaller sample through the column (as in the drip method), total protein recovery typically increased by 10 to 20%.

However, gravity-flow protocols typically require a significant chase buffer volume to be added after the sample has entered the resin bed. This is done to simplify sample collection and maximize protein recovery. For example, following the drip protocol for the PD-10 columns, we collected the 1.5mL sample in fractions totaling 3.5mL. This resulted in a significant (2.3-fold) dilution of the sample. The combined effect of protein loss and sample dilution can be seen by comparing SDS-PAGE band intensities resulting from equal load volumes of the samples recovered from each column tested (Figure 3). Overall protein recovery as assessed by this metric was best for the Zeba Spin Desalting Columns.

Figure 3. Better overall performance with Zeba Spin Desalting Columns. Thermo Scientific Zeba Columns provide higher protein recovery and less sample dilution over a wider range of sample concentrations and volume compared to alternative products. Zeba Spin Desalting Columns (7K MWCO, 10mL) and Disposable PD-10 Desalting Columns (GE Healthcare) were used to desalt 1.5, 2.5 and 3.5mL samples of bovine serum albumin (BSA) at concentrations of 0.04, 0.2, and 1mg/mL. Desalting was performed according to each manufacturer’s recommended protocols for either spin (centrifuge) or drip (gravity) procedures. Three sets of columns were equilibrated in final buffer and then loaded with 1.5, 2.5, and 3.5mL samples. For each electrophoresis gel, an aliquot of starting sample equal to 1µg of BSA was loaded in Lane 1 as the Load Control; all other desalted samples were loaded in the gel at the same volume as the Load Control. Differences in intensity between lanes are a combination of protein recovery and sample dilution caused by desalting.

The exact efficiency of buffer-exchange and protein recovery that can be obtained with any gel filtration resin and method depends upon a variety of factors, including the individual characteristics of the molecules involved, such as shape, hydrophobicity, solubility, binding sites and concentration. Each sample should be examined and optimized independently. Nevertheless, our experiments with pure and complex protein mixtures in Tris-buffered saline, suggest that Zeba Spin Desalting Columns will perform as well or better than the GE Healthcare and Bio-Rad columns in typical applications with protein samples.

Conclusions

Several manufacturers offer spin desalting columns with 5K- to 7K-MWCO resins for rapid processing of 0.01mL to 4mL protein samples. We tested similarly sized GE Healthcare, Bio-Rad and Thermo Scientific Zeba columns for desalting performance (efficiency of salt removal and protein recovery). Although the efficiency of salt removal was comparable for all columns (>95%), protein recovery was significantly greater with Zeba Spin Desalting Columns than with the competing products.

Overall, Zeba Columns provided the best protein recovery (with minimal or no sample dilution) for the widest range of sample volumes and concentrations tested. The five sizes of ready-to-use Zeba Spin Desalting Columns are compatible with many standard laboratory instruments (microcentrifuges and centrifuges) and consumables (collection tubes) to accommodate sample volumes between 2µL and 4mL.

Methods

The following products were tested with sample volumes of 40, 80 and 120µL:

Solutions of bovine serum albumin (BSA) and HeLa cell extracts were prepared at concentrations of 0.04, 0.2, and 1mg/mL in 25mM Tris, 500mM NaCl, pH 7.5. Columns were equilibrated with a buffer of 25mM Tris, 25mM NaCl, pH 7.5. Columns were used according to respective manufacturers’ recommended protocols. For all spin columns, the samples were processed at the indicated volumes with no stack or chase buffer added. For the PD-10 column (GE Healthcare), both spin and gravity-flow (drip) protocols were tested. For the drip protocol (and per the manufacturer’s instructions), samples were loaded to the columns and then collected with a chase buffer volume of 3.5mL regardless of the initial sample volume.

Efficiency of salt removal (desalting, per se) from 500mM NaCl to 25mM NaCl was determined by measuring the conductivity (mS/cm) of the starting and final samples compared to a standard curve.

Protein recovery was calculated by measuring the protein concentration of the desalted sample and multiplying this value by the recovered volume. Protein concentrations were determined with the BCA Protein Assay Kit (Part No.23227).

Samples were also analyzed by SDS-PAGE. For SDS-PAGE the initial sample was loaded at 1µg protein. All other samples were loaded using this same volume. As such, differences in intensity between lanes are a combination of differences in protein recovery and volumetric dilution factor.

Fast—no fraction screening or waiting for protein to emerge by gravity-flow

Easy-to-use—no cumbersome column preparation or equilibration
Flexible—available in spin columns, filter spin plates and cartridges for a range of needs

Economical—great performance at lower cost than other commercially available cartridges

Even very dilute (25 µg/mL) protein samples can be successfully processed to obtain greater than 95% retention (removal) of salts and other small molecules (< 1000 MW) and good recovery of proteins and other macromolecules (> 7000 MW).